Composition and method for diagnosing auto-immune hepatitis

ABSTRACT

A composition and method for the diagnosis of autoimmune hepatitis. The composition, which contains SLA antigens detects soluble liver antigen (SLA) auto-antibodies, which occur in sera of patients suffering from chronic hepatitis.

This is a division of application Ser. No. 09/249,020 filed Feb. 12,1999.

BACKGROUND OF THE INVENTION

The present invention relates to a composition and method for diagnosingauto-immune hepatitis, which use an immune reaction to detect solubleliver antigen (“SLA”) auto-antibodies.

Auto-immune hepatitis (AIH) is a chronic inflammation of the liver thatwhen left untreated leads to cirrhosis, but when treated has a very goodprognosis. For this reason, a timely diagnosis is important. It isestimated that 5% of all patients in Western countries who have chronichepatitis have AIH. At the present time, there is no specific diagnostictest for AIH. Rather the diagnosis for AIH is undertaken by a pluralityof diagnosis, such as excluding viral hepatitis, recognizing [hyper]immunoglobulinaemia, recognizing the tissue type (HLA type), anddetecting auto-antibodies. Auto-antibodies are found in about 90% ofpatients with chronic hepatitis, and most of the detectableauto-antibodies are also present, at least in low titers, in otherinflammations of the liver as well. In particular, these auto-antibodiesare antibodies to nuclear antigens (ANA) and unstriated muscles (SMA),as well as the very rare antibodies to cytochrome p450 (LKM). SLAauto-antibodies were described for the first time in 1987 (Manns M. etal., Lancet 1987;1:292-4). Tests have shown that SLA auto-antibodiesoccur in about 25 to 30% of patients having AIH, but hardly ever occurin patients having other diseases, including other auto-immune diseases(Lohse A. W. et al., Z. Gastroenterol 1995;33:527-33). Detecting SLAauto-antibodies therefore provides a significant diagnostic procedurefor recognizing AIH.

The present invention is directed to use and detection of SLA antigens,which are a prerequisite for developing a specific immunoassay fordetecting SLA auto-antibodies in patients' sera. Previous methods knownin the art could not detect such SLA auto-antibodies in patients' sera.Liver cytokeratins 8 and 18 were described in 1990 as target antigens ofthe SLA auto-antibodies (Journal of Hepatology, 1990; 11: 232 to 239),however, these findings were never confirmed, and were even refuted(Wies I. et al., Z. Gastroenterol. 1998;36:93).

SUMMARY OF THE INVENTION

The invention is directed to a composition for detecting the presence ofSLA auto-antibodies in blood serum, said composition comprising anantigen that is recognized by SLA auto-antibodies. In particular theinvention is directed to antigens having amino acid sequence SEQ IDNO:2, SEQ ID NO:4, or antigenic derivatives thereof, or encoded by DNAhaving DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivativesthereof. Another embodiment of the invention is a purified protein orpolypeptide recognized by SLA auto-antibodies. In a preferred embodimentof the invention the purified protein has the amino acid sequence SEQ IDNO:2, SEQ ID NO:4, or antigenic derivatives thereof, or encoded by DNAhaving DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivativesthereof, or is a fusion protein containing either SEQ ID NO:2, SEQ IDNO:4, or antigenic derivatives thereof, or encoded by DNA having DNAsequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivatives thereof. Inanother aspect of the invention, there is disclosed a cDNA having anuclcotide sequence that codes for an antigen recognized by SLAauto-antibodies in blood serum. In a preferred embodiment of this aspectof the invention the cDNA codes for an antigen having the amino acidsequence SEQ ID NO:2, SEQ ID NO:4, or antigenic derivatives thereof, orhas DNA sequence SEQ ID NO:1, SEQ ID NO:3, or antigenic derivativesthereof.

Another aspect of the invention is directed to a method of detecting thepresence of SLA auto-antibodies in a blood sample by binding thecomposition of the invention to a matrix, detecting the binding of SLAauto-antibodies bound to the antigens in the composition, andcorrelating such binding to the presence of SLA auto-antibodies in thesample. Examples of suitable methods in which the present compositionscan be used to detect SLA auto-antibodies in blood include immunoassayssuch as, but not limited to, radioimmunoassay, chemiluminescence,immunoassay, immunoblot assay, enzyme assay and inhibition immunoassay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a composition and a method fordiagnosing auto-immune hepatitis, by enabling one to detect in the bloodserum of a patient, antibodies to two proteins or polypeptides (SLAantigens), which antigens wholly or partially contain the amino acidsequence corresponding to SEQ ID NO:2, SEQ ID NO:4, or antigenicderivatives thereof, or encoded by DNA having DNA sequence SEQ ID NO:1,SEQ ID NO:3, or antigenic derivatives thereof of the SLA antigens, whichare recognized by SLA auto-antibodies. These polypeptides are referredto as SLA-1 and as SLA-2, One embodiment of the invention is directed tosynthetic or natural variants of SLA-1 or SLA-2, or synthetic or naturalvariants of partial or incomplete polypeptides of SLA-1 or SLA-2, whichcorrespond wholly or partially to the amino acid sequences or antigenicderivatives thereof and are likewise recognized by SLA auto-antibodies.

Another embodiment of the invention is directed to cDNA, which encodes anatural or synthetic variant of one of the SLA antigens SLA-1 or SLA-2,having a nucleotide sequence corresponding to SEQ ID NO:1 or to SEQ IDNO:3 or to antigenic derivatives thereof. The cDNAs are present as twospliced variants, the longer of these (SEQ ID NO:3 having an insertionof 156 nucliotides. SLA-1 and SLA-2 have similar nucleotide sequences,except that SLA-2 contains a 156 nuclcotide insert.

SLA-positive sera, i.e. SLA auto-antibody containing sera, produces adouble band on a Western blot when probed with the SLA antigens. Thedouble band measures 50 kDa, which corresponds to the molecular weightof the SLA antigens, SLA-1 and SLA-2. When SLA-positive serum isincubated with the fusion protein according to the present invention,e.g. SLA-1 or SLA-2 fused to another protein or polypeptide, the doubleband corresponding to the SLA-antigen is not detected. Incubation ofSLA-positive sera with a control fusion protein, which does not containeither SLA-1 or SLA-2 does not eliminate the appearance of a double bandon Western blots.

The cDNA according to the present invention allows for the production oflarge quantities of the SLA antigens in a recombinant fashion. Further,the concentration of SLA antigens can be measured using known SLAauto-antibody methods.

In another aspect of the invention there is disclosed a method fordetermining the presence of SLA auto-antibodies in a blood sample usingthe composition of the invention in known assay methods such as, but notlimited to, radioimmunoassay (RIA), chemiluminescence immunoassay (CIA),immunoblot assay or enzyme immunoassay (EIA). In these assays, one ofthe SLA antigens (SLA-1 or SLA-2) is bound to a matrix, such as amicrotiter plate or a blot membrane. Then the patient's serum to betested is applied to the matrix with the bound SLA antigen. Followingincubation, the test serum is washed off, The specific binding of SLAauto-antibodies (in the test serum) to the matrix-bound SLA-antigen isdetected and verified by using a secondary antibody (anti-humanimmunoglobulin or an immunoglobulin subclass, for example) that has beenlabeled with a tracer. Tracers commonly used include enzyme compoundssuch as peroxidase or alkaline phosphiatase, radioactive compounds, or achemiluminescing substances. The more SLA auto-antibodies present in thetest serum, the more tracer is bound to the matrix. Normal serum fromhealthy blood donors can be used as a negative control. Additionalcontrols include the use of SLA auto-antibody positive serum that hasSLA auto-antibodies present at known various concentrations.

Alternatively, an inhibition immunoassay can also be performed using theSLA antigens of the present invention. In such an assay the SLA antigensbind to a selected SLA auto-antibody, which has been labeled with atracer. The amount of binding of the labeled SLA auto-antibody to theSLA antigens is measured as the control. A patient's sera is then testedagainst the control. The patient's serum is added along with the labeledSLA auto-antibodies. SLA auto-antibodies present in the patient's serumcompete with the labeled SLA auto-antibodies in binding to the SLAantigens. Thus, if the patient's serum contains SLA auto-antibodies, theamount of labeled SLA auto-antibodies present will be less than thecontrol. Therefore the detection of fewer bound labeled SLAauto-antibodies reveals the presence of competing SLA auto-antibodies inthe test serum.

EXAMPLE 1 Detecting SLA Auto-antibodies in the ELISA Test Using theRecombinant SLA Antigen (SEQ ID No:2)

The recombinant SLA antigen was purified using the his-tag. 50 μg of theSLA antigen in 50 μl phosphate buffered saline (“PBS”)(pH 7.0) wasplaced in a microtiter plate, and left over night. After pipetting offand washing the microtiter plate, a post-coating was carried out for onehour using a 1% Bovine Serum Albumin (“BSA”)/PBS solution at roomtemperature. 50 μl each of normal sera, patients' sera, and control serawere diluted 1:100 with PBS. 50 μl of each diluted serum was incubatedfor 30 minutes at room temperature on its respective microtiter plate.The plates were then washed 1-5 times and a peroxidase-labeled secondaryantibody, an antihuman immunoglobulin, diluted 1:8000 was then added andallowed to incubate. After washing out the excess secondary antibody,ABTS solution [55 mg of 2,2′-azidodi-3-ethyl-benzthiazine-6-sulphonicacid (Serva, Heidelberg) in 5 ml 0.01 M K₂HPO₄ (pH 6.0), containing 50μl H₂O₂, diluted in a 1:300 proportion in PBS] was added. The opticaldensity of the microtiter plates was measured with a Titertec MultiscanMC (Flow Laboratories, Meckenheim). Table 1 reveals that all of thepatients' sera containing SLA auto-bodies exhibited distinctly elevatedvalues of light absorption, as compared to normal serum, and serum ofpatients who suffered from other liver diseases.

TABLE 1 Serum OD normal serum 1 0.476 normal serum 1 0.471 SLA-serum 11.863 SLA-serum 2 1.995 SLA-serum 3 1.791 SLA-serum 4 1.867 AMA-pos.serum (PBC) 0.615 LKM-pos. serum (hep. C) 0.769 Explanations: OD =optical density (extinction); AMA = anti-mitochondrial antibody; PBC =primary biliary cirrhosis; LKM = liver-kidney-microsomal; BSA = bovineserum albumin; PBS = phosphate-buffered saline

4 1 1335 DNA Homo sapiens CDS (1)...(1335) 1 tcg cgg cgg gag agc ggc tggtgt cgc cgg ctt acg tgc ggc agg gct 48 Ser Arg Arg Glu Ser Gly Trp CysArg Arg Leu Thr Cys Gly Arg Ala 1 5 10 15 gtg agg ccc gcc gct cgc atgagc acc tca tac ggc tgc ttc tgg aga 96 Val Arg Pro Ala Ala Arg Met SerThr Ser Tyr Gly Cys Phe Trp Arg 20 25 30 agg ttc att cat ggc att gga cgatcc ggt gat att tct gct gtg caa 144 Arg Phe Ile His Gly Ile Gly Arg SerGly Asp Ile Ser Ala Val Gln 35 40 45 cca aaa gct gca ggc tct agc ctt ttgaac aaa att acc aat tct ttg 192 Pro Lys Ala Ala Gly Ser Ser Leu Leu AsnLys Ile Thr Asn Ser Leu 50 55 60 gtc ctg gac att ata aag ctg gct ggt gtccat aca gta gcc aac tgc 240 Val Leu Asp Ile Ile Lys Leu Ala Gly Val HisThr Val Ala Asn Cys 65 70 75 80 ttt gta gtt cct atg gca act ggt atg agtcta act ctg tgt ttc tta 288 Phe Val Val Pro Met Ala Thr Gly Met Ser LeuThr Leu Cys Phe Leu 85 90 95 aca tta cga cac aaa aga cca aag gca aag tatatt ata tgg cca cga 336 Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr IleIle Trp Pro Arg 100 105 110 ata gac cag aag tcc tgc ttt aaa tcc atg atcact gca ggt ttt gag 384 Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile ThrAla Gly Phe Glu 115 120 125 cct gtg gtg ata gaa aat gtt ttg gaa ggt gacgag ctg cgt aca gac 432 Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp GluLeu Arg Thr Asp 130 135 140 ctg aaa gca gtg gag gct aaa gtc cag gaa cttggg cct gat tgc att 480 Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu GlyPro Asp Cys Ile 145 150 155 160 ctg tgt att cat tct act aca tcc tgt tttgct cca agg gtg cct gat 528 Leu Cys Ile His Ser Thr Thr Ser Cys Phe AlaPro Arg Val Pro Asp 165 170 175 aga tta gaa gaa ctg gct gtg att tgt gctaat tat gac att cca cat 576 Arg Leu Glu Glu Leu Ala Val Ile Cys Ala AsnTyr Asp Ile Pro His 180 185 190 ata gtt aat aat gct tat gga gtg cag tcttca aag tgt atg cat ctc 624 Ile Val Asn Asn Ala Tyr Gly Val Gln Ser SerLys Cys Met His Leu 195 200 205 att cag cag ggg gct cga gtt ggt aga atagat gct ttt gtt cag agc 672 Ile Gln Gln Gly Ala Arg Val Gly Arg Ile AspAla Phe Val Gln Ser 210 215 220 ttg gac aaa aat ttt atg gtt cca gta ggtggt gct ata att gct ggc 720 Leu Asp Lys Asn Phe Met Val Pro Val Gly GlyAla Ile Ile Ala Gly 225 230 235 240 ttt aat gat tca ttc att cag gaa atcagc aag atg tat cca gga aga 768 Phe Asn Asp Ser Phe Ile Gln Glu Ile SerLys Met Tyr Pro Gly Arg 245 250 255 gct tca gct tca cct tct tta gat gtcctt att act tta ttg tca ctt 816 Ala Ser Ala Ser Pro Ser Leu Asp Val LeuIle Thr Leu Leu Ser Leu 260 265 270 gga tca aat ggc tat aag aag cta ctaaaa gaa aga aag gaa atg ttt 864 Gly Ser Asn Gly Tyr Lys Lys Leu Leu LysGlu Arg Lys Glu Met Phe 275 280 285 tca tat ttg tcc aac caa ata aag aagttg tca gaa gcc tac aat gaa 912 Ser Tyr Leu Ser Asn Gln Ile Lys Lys LeuSer Glu Ala Tyr Asn Glu 290 295 300 aga ctg ttg cat aca cct cac aat cccata tct tta gct atg aca ctt 960 Arg Leu Leu His Thr Pro His Asn Pro IleSer Leu Ala Met Thr Leu 305 310 315 320 aaa aca cta gat gaa cac cgt gacaaa gct gtc act cag ctt ggc tcg 1008 Lys Thr Leu Asp Glu His Arg Asp LysAla Val Thr Gln Leu Gly Ser 325 330 335 atg ctt ttt acc aaa cag gtt tctgga gcc agg gtt gtg cct ctt ggg 1056 Met Leu Phe Thr Lys Gln Val Ser GlyAla Arg Val Val Pro Leu Gly 340 345 350 tcc atg caa act gtg agt ggc tatact ttc aga ggc ttt atg tca cat 1104 Ser Met Gln Thr Val Ser Gly Tyr ThrPhe Arg Gly Phe Met Ser His 355 360 365 aca aat aat tac cct tgt gct tacctc aat gct gca tca gcc atc gga 1152 Thr Asn Asn Tyr Pro Cys Ala Tyr LeuAsn Ala Ala Ser Ala Ile Gly 370 375 380 atg aar atg cag gat gtg gac ctgttc ata aac ara ctt gac agg tgt 1200 Met Lys Met Gln Asp Val Asp Leu PheIle Asn Xaa Leu Asp Arg Cys 385 390 395 400 tta aag gca gta aga aaa gaacga agt aaa gag agt gat gac aat tat 1248 Leu Lys Ala Val Arg Lys Glu ArgSer Lys Glu Ser Asp Asp Asn Tyr 405 410 415 gac aaa act gaa rat gtg gatatt gaa gaa atg gct tta aaa cta gat 1296 Asp Lys Thr Glu Xaa Val Asp IleGlu Glu Met Ala Leu Lys Leu Asp 420 425 430 aat gta ctt ctt gac aca taccag gat gct tct tca tga 1335 Asn Val Leu Leu Asp Thr Tyr Gln Asp Ala SerSer * 435 440 2 444 PRT Homo sapiens VARIANT (1)...(444) Xaa = Any AminoAcid 2 Ser Arg Arg Glu Ser Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 15 10 15 Val Arg Pro Ala Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg20 25 30 Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile Ser Ala Val Gln35 40 45 Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile Thr Asn Ser Leu50 55 60 Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr Val Ala Asn Cys65 70 75 80 Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr Leu Cys PheLeu 85 90 95 Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile Ile Trp ProArg 100 105 110 Ile Asp Gln Lys Ser Cys Phe Lys Ser Met Ile Thr Ala GlyPhe Glu 115 120 125 Pro Val Val Ile Glu Asn Val Leu Glu Gly Asp Glu LeuArg Thr Asp 130 135 140 Leu Lys Ala Val Glu Ala Lys Val Gln Glu Leu GlyPro Asp Cys Ile 145 150 155 160 Leu Cys Ile His Ser Thr Thr Ser Cys PheAla Pro Arg Val Pro Asp 165 170 175 Arg Leu Glu Glu Leu Ala Val Ile CysAla Asn Tyr Asp Ile Pro His 180 185 190 Ile Val Asn Asn Ala Tyr Gly ValGln Ser Ser Lys Cys Met His Leu 195 200 205 Ile Gln Gln Gly Ala Arg ValGly Arg Ile Asp Ala Phe Val Gln Ser 210 215 220 Leu Asp Lys Asn Phe MetVal Pro Val Gly Gly Ala Ile Ile Ala Gly 225 230 235 240 Phe Asn Asp SerPhe Ile Gln Glu Ile Ser Lys Met Tyr Pro Gly Arg 245 250 255 Ala Ser AlaSer Pro Ser Leu Asp Val Leu Ile Thr Leu Leu Ser Leu 260 265 270 Gly SerAsn Gly Tyr Lys Lys Leu Leu Lys Glu Arg Lys Glu Met Phe 275 280 285 SerTyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu Ala Tyr Asn Glu 290 295 300Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu Ala Met Thr Leu 305 310315 320 Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr Gln Leu Gly Ser325 330 335 Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val Val Pro LeuGly 340 345 350 Ser Met Gln Thr Val Ser Gly Tyr Thr Phe Arg Gly Phe MetSer His 355 360 365 Thr Asn Asn Tyr Pro Cys Ala Tyr Leu Asn Ala Ala SerAla Ile Gly 370 375 380 Met Lys Met Gln Asp Val Asp Leu Phe Ile Asn XaaLeu Asp Arg Cys 385 390 395 400 Leu Lys Ala Val Arg Lys Glu Arg Ser LysGlu Ser Asp Asp Asn Tyr 405 410 415 Asp Lys Thr Glu Xaa Val Asp Ile GluGlu Met Ala Leu Lys Leu Asp 420 425 430 Asn Val Leu Leu Asp Thr Tyr GlnAsp Ala Ser Ser 435 440 3 1491 DNA Homo sapiens CDS (1)...(1491)misc_feature (1)...(1491) n = A,T,C or G 3 tcg cgg cgg gag agc ggc tggtgt cgc cgg ctt acg tgc ggc agg gct 48 Ser Arg Arg Glu Ser Gly Trp CysArg Arg Leu Thr Cys Gly Arg Ala 1 5 10 15 gtg agg ccc gcc gct cgc atgagc acc tca tac ggc tgc ttc tgg aga 96 Val Arg Pro Ala Ala Arg Met SerThr Ser Tyr Gly Cys Phe Trp Arg 20 25 30 agg gcw nnn tgt cca aag aat ggctgg gat gaa agt aca ctt gaa ctc 144 Arg Xaa Xaa Cys Pro Lys Asn Gly TrpAsp Glu Ser Thr Leu Glu Leu 35 40 45 ttt tta cat gaa ctt gca atc atg gacagc aac aat ttc tta ggc aat 192 Phe Leu His Glu Leu Ala Ile Met Asp SerAsn Asn Phe Leu Gly Asn 50 55 60 tgt ggt gtg gga gaa agg gaa ggg aga gtggca tcc gca ctg gtt gct 240 Cys Gly Val Gly Glu Arg Glu Gly Arg Val AlaSer Ala Leu Val Ala 65 70 75 80 cgt cgt cat tac agg ttc att cat ggc attgga cga tcc ggt gat att 288 Arg Arg His Tyr Arg Phe Ile His Gly Ile GlyArg Ser Gly Asp Ile 85 90 95 tct gct gtg caa cca aaa gct gca ggc tct agcctt ttg aac aaa att 336 Ser Ala Val Gln Pro Lys Ala Ala Gly Ser Ser LeuLeu Asn Lys Ile 100 105 110 acc aat tct ttg gtc ctg gac att ata aag ctggct ggt gtc cat aca 384 Thr Asn Ser Leu Val Leu Asp Ile Ile Lys Leu AlaGly Val His Thr 115 120 125 gta gcc aac tgc ttt gta gtt cct atg gca actggt atg agt cta act 432 Val Ala Asn Cys Phe Val Val Pro Met Ala Thr GlyMet Ser Leu Thr 130 135 140 ctg tgt ttc tta aca tta cga cac aaa aga ccaaag gca aag tat att 480 Leu Cys Phe Leu Thr Leu Arg His Lys Arg Pro LysAla Lys Tyr Ile 145 150 155 160 ata tgg cca cga ata gac cag aag tcc tgcttt aaa tcc atg atc act 528 Ile Trp Pro Arg Ile Asp Gln Lys Ser Cys PheLys Ser Met Ile Thr 165 170 175 gca ggt ttt gag cct gtg gtg ata gaa aatgtt ttg gaa ggt gac gag 576 Ala Gly Phe Glu Pro Val Val Ile Glu Asn ValLeu Glu Gly Asp Glu 180 185 190 ctg cgt aca gac ctg aaa gca gtg gag gctaaa gtc cag gaa ctt ggg 624 Leu Arg Thr Asp Leu Lys Ala Val Glu Ala LysVal Gln Glu Leu Gly 195 200 205 cct gat tgc att ctg tgt att cat tct actaca tcc tgt ttt gct cca 672 Pro Asp Cys Ile Leu Cys Ile His Ser Thr ThrSer Cys Phe Ala Pro 210 215 220 agg gtg cct gat aga tta gaa gaa ctg gctgtg att tgt gct aat tat 720 Arg Val Pro Asp Arg Leu Glu Glu Leu Ala ValIle Cys Ala Asn Tyr 225 230 235 240 gac att cca cat ata gtt aat aat gcttat gga gtg cag tct tca aag 768 Asp Ile Pro His Ile Val Asn Asn Ala TyrGly Val Gln Ser Ser Lys 245 250 255 tgt atg cat ctc att cag cag ggg gctcga gtt ggt aga ata gat gct 816 Cys Met His Leu Ile Gln Gln Gly Ala ArgVal Gly Arg Ile Asp Ala 260 265 270 ttt gtt cag agc ttg gac aaa aat tttatg gtt cca gta ggt ggt gct 864 Phe Val Gln Ser Leu Asp Lys Asn Phe MetVal Pro Val Gly Gly Ala 275 280 285 ata att gct ggc ttt aat gat tca ttcatt cag gaa atc agc aag atg 912 Ile Ile Ala Gly Phe Asn Asp Ser Phe IleGln Glu Ile Ser Lys Met 290 295 300 tat cca gga aga gct tca gct tca ccttct tta gat gtc ctt att act 960 Tyr Pro Gly Arg Ala Ser Ala Ser Pro SerLeu Asp Val Leu Ile Thr 305 310 315 320 tta ttg tca ctt gga tca aat ggctat aag aag cta cta aaa gaa aga 1008 Leu Leu Ser Leu Gly Ser Asn Gly TyrLys Lys Leu Leu Lys Glu Arg 325 330 335 aag gaa atg ttt tca tat ttg tccaac caa ata aag aag ttg tca gaa 1056 Lys Glu Met Phe Ser Tyr Leu Ser AsnGln Ile Lys Lys Leu Ser Glu 340 345 350 gcc tac aat gaa aga ctg ttg cataca cct cac aat ccc ata tct tta 1104 Ala Tyr Asn Glu Arg Leu Leu His ThrPro His Asn Pro Ile Ser Leu 355 360 365 gct atg aca ctt aaa aca cta gatgaa cac cgt gac aaa gct gtc act 1152 Ala Met Thr Leu Lys Thr Leu Asp GluHis Arg Asp Lys Ala Val Thr 370 375 380 cag ctt ggc tcg atg ctt ttt accaaa cag gtt tct gga gcc agg gtt 1200 Gln Leu Gly Ser Met Leu Phe Thr LysGln Val Ser Gly Ala Arg Val 385 390 395 400 gtg cct ctt ggg tcc atg caaact gtg agt ggc tat act ttc aga ggc 1248 Val Pro Leu Gly Ser Met Gln ThrVal Ser Gly Tyr Thr Phe Arg Gly 405 410 415 ttt atg tca cat aca aat aattac cct tgt gct tac ctc aat gct gca 1296 Phe Met Ser His Thr Asn Asn TyrPro Cys Ala Tyr Leu Asn Ala Ala 420 425 430 tca gcc atc gga atg aar atgcag gat gtg gac ctg ttc ata aac ara 1344 Ser Ala Ile Gly Met Lys Met GlnAsp Val Asp Leu Phe Ile Asn Xaa 435 440 445 ctt gac agg tgt tta aag gcagta aga aaa gaa cga agt aaa gag agt 1392 Leu Asp Arg Cys Leu Lys Ala ValArg Lys Glu Arg Ser Lys Glu Ser 450 455 460 gat gac aat tat gac aaa actgaa rat gtg gat att gaa gaa atg gct 1440 Asp Asp Asn Tyr Asp Lys Thr GluXaa Val Asp Ile Glu Glu Met Ala 465 470 475 480 tta aaa cta gat aat gtactt ctt gac aca tac cag gat gct tct tca 1488 Leu Lys Leu Asp Asn Val LeuLeu Asp Thr Tyr Gln Asp Ala Ser Ser 485 490 495 tga 1491 * 4 496 PRTHomo sapiens VARIANT (1)...(496) Xaa = Any Amino Acid 4 Ser Arg Arg GluSer Gly Trp Cys Arg Arg Leu Thr Cys Gly Arg Ala 1 5 10 15 Val Arg ProAla Ala Arg Met Ser Thr Ser Tyr Gly Cys Phe Trp Arg 20 25 30 Arg Xaa XaaCys Pro Lys Asn Gly Trp Asp Glu Ser Thr Leu Glu Leu 35 40 45 Phe Leu HisGlu Leu Ala Ile Met Asp Ser Asn Asn Phe Leu Gly Asn 50 55 60 Cys Gly ValGly Glu Arg Glu Gly Arg Val Ala Ser Ala Leu Val Ala 65 70 75 80 Arg ArgHis Tyr Arg Phe Ile His Gly Ile Gly Arg Ser Gly Asp Ile 85 90 95 Ser AlaVal Gln Pro Lys Ala Ala Gly Ser Ser Leu Leu Asn Lys Ile 100 105 110 ThrAsn Ser Leu Val Leu Asp Ile Ile Lys Leu Ala Gly Val His Thr 115 120 125Val Ala Asn Cys Phe Val Val Pro Met Ala Thr Gly Met Ser Leu Thr 130 135140 Leu Cys Phe Leu Thr Leu Arg His Lys Arg Pro Lys Ala Lys Tyr Ile 145150 155 160 Ile Trp Pro Arg Ile Asp Gln Lys Ser Cys Phe Lys Ser Met IleThr 165 170 175 Ala Gly Phe Glu Pro Val Val Ile Glu Asn Val Leu Glu GlyAsp Glu 180 185 190 Leu Arg Thr Asp Leu Lys Ala Val Glu Ala Lys Val GlnGlu Leu Gly 195 200 205 Pro Asp Cys Ile Leu Cys Ile His Ser Thr Thr SerCys Phe Ala Pro 210 215 220 Arg Val Pro Asp Arg Leu Glu Glu Leu Ala ValIle Cys Ala Asn Tyr 225 230 235 240 Asp Ile Pro His Ile Val Asn Asn AlaTyr Gly Val Gln Ser Ser Lys 245 250 255 Cys Met His Leu Ile Gln Gln GlyAla Arg Val Gly Arg Ile Asp Ala 260 265 270 Phe Val Gln Ser Leu Asp LysAsn Phe Met Val Pro Val Gly Gly Ala 275 280 285 Ile Ile Ala Gly Phe AsnAsp Ser Phe Ile Gln Glu Ile Ser Lys Met 290 295 300 Tyr Pro Gly Arg AlaSer Ala Ser Pro Ser Leu Asp Val Leu Ile Thr 305 310 315 320 Leu Leu SerLeu Gly Ser Asn Gly Tyr Lys Lys Leu Leu Lys Glu Arg 325 330 335 Lys GluMet Phe Ser Tyr Leu Ser Asn Gln Ile Lys Lys Leu Ser Glu 340 345 350 AlaTyr Asn Glu Arg Leu Leu His Thr Pro His Asn Pro Ile Ser Leu 355 360 365Ala Met Thr Leu Lys Thr Leu Asp Glu His Arg Asp Lys Ala Val Thr 370 375380 Gln Leu Gly Ser Met Leu Phe Thr Lys Gln Val Ser Gly Ala Arg Val 385390 395 400 Val Pro Leu Gly Ser Met Gln Thr Val Ser Gly Tyr Thr Phe ArgGly 405 410 415 Phe Met Ser His Thr Asn Asn Tyr Pro Cys Ala Tyr Leu AsnAla Ala 420 425 430 Ser Ala Ile Gly Met Lys Met Gln Asp Val Asp Leu PheIle Asn Xaa 435 440 445 Leu Asp Arg Cys Leu Lys Ala Val Arg Lys Glu ArgSer Lys Glu Ser 450 455 460 Asp Asp Asn Tyr Asp Lys Thr Glu Xaa Val AspIle Glu Glu Met Ala 465 470 475 480 Leu Lys Leu Asp Asn Val Leu Leu AspThr Tyr Gln Asp Ala Ser Ser 485 490 495

I claim:
 1. A cDNA comprising a nucleotide sequence that codes for anantigen recognized by soluble liver antigen (SLA) auto antibodies inblood serum wherein said cDNA codes for an antigen having the amino acidsequence SEQ ID NO:2 or SEQ ID NO:4.
 2. The cDNA of claim 1 wherein saidcDNA codes for an antigen having the amino acid sequence SEQ ID NO:2. 3.The cDNA of claim 1 wherein said cDNA codes for an antigen having theamino acid sequence SEQ ID NO:4.
 4. A cDNA comprising DNA sequence SEQID NO:1.
 5. A cDNA comprising DNA sequence SEQ ID NO:3.